Method for producing a correctly folded, biological active recombinant protein

ABSTRACT

Methods for producing correctly folded biological active recombinant protein or polypeptide comprise the steps of expressing the protein or polypeptide in prokaryotic cells, harvesting the cells, directly solubilizing the cells in a buffer at pH of about 8 to 11 with the chaotropic agent and a reducing agent, and diluting the solution with water and a diluent. The method is conducted in the absence of mechanical disruption of the cells and isolation and washing of refractile bodies.

The present invention relates to a method for producing a correctlyfolded, biological active recombinant protein or polypeptide, comprisingthe steps of expression of the protein in prokaryotic cells, harvest ofthe cells, direct solubilization of the cells in a buffer at pH about 8to 11 and thereafter dilution with water and a diluent.

The protein or polypeptide is preferably GH, IGF-I or IGF-II.

INTRODUCTION

A general, major problem when recombinant proteins are overproduced inefficient bacterial expression systems is related to the folding of theprotein products into their native conformations. Many high levelexpression systems in Escherichia coli result in the production ofaggregates of denatured proteins, so called inclusion bodies, which insome cases may be refolded into the wanted native protein. Generalmethods to facilitate and render the refolding effective have beenfound. One is the use of a class of heat-shock-proteins (HSP) and theother is folding-enzymes. By using HSP, aggregation is avoided and byusing the folding enzymes, the speed of refolding is accelerated.

However, not all proteins are susceptible for these methods and othersolutions to enhance refolding yields have been suggested. In an articleby J D Carlson et al in Biotechnology, Vol 10, January 1992, the use ofmonoclonal antibodies during protein refolding, to enhance the yield ofnative protein, especially S-Protein, has been disclosed.

Another suggested method for the recovery of the native protein issolubilization of the inclusion body protein with a denaturant, such asguanidine or urea and if needed a reduction of the disulphide bond. Bydilution or dialysis and reoxidation, the protein can be refolded to thenative protein.

Successful refolding, without formation of new inclusion bodies, isgenerally difficult at high concentrations of the recombinant protein.The best yield is generally achieved at concentrations around 20-200μg/ml. Refolding is therefore considered to be a very expensiveproduction form that demands a cost intensive drug.

However, the yield of a refolding procedure is unpredictable since theprotein product often aggregates or gets modified. In addition, forIGF-I and II, the soluble refolded fraction will contain misfoldedspecies and the overall yield of correctly folded growth factor israther low (Samuelsson, E., et al (1991) Bio/Technology Vol. 9, Page363).

In order to increase the yield of correctly folded IGF-I differentmethods have been proposed.

Human insulin-like growth factor I (IGF-I) is a single-chain peptidegrowth factor of 70 amino acids, originally isolated from serum. IGF-Iis positively regulated by growth hormone (GH) and shows mitogeniceffects on many cell types. Therefore, IGF-I is thought to mediate manyof the growth promoting effects of GH. In the regions of homology, IGF-Iand insulin are 49% homologous, including the six cysteine residues,furnishing three disulphide bridges. The three dimensional structure ofIGF-I has been modelled based on the x-ray structure of insulin, andthis model has recently been confirmed in the disulphide bridge regionsby distance constraints obtained by 2-D NMR spectroscopy of IGF-I (for areview on IGF, see: Insulin-like growth factors I and II, Humbel R. E,Eur. J. Biochem 190, 445462,1990).

Human recombinant IGF-I has been produced as a secreted product in bothEscherichia coli and Saccharomyces cerevisiae. In isolated material fromboth species, IGF-I is found mainly as miss-folded forms withintermolecular disulphides. In addition, in vitro refolding of reducedIGF-I in the presence of oxygen, has demonstrated that native,miss-matched and aggregated IGF-I accumulate, even under diluterefolding conditions.

The refolding yield of recombinant IGF-I was significantly improved byutilising a fused fusion partner, consisting of two IgG-binding domains(ZZ) derived from staphylococcal protein A (Samuelsson, E., et al (1991)Bio/Technology Vol. 9, Page 363). The ZZ fusion partner is used tosolubilise misfolded molecules before, during and after reduction andreoxidation. The yield of correctly folded IGF-I is shown to besubstantially increased but there is still a significant amount ofmisfolded IGF.

Patents and patent applications have also described the problem ofmisfolded IGF and suggested different improvements.

WO 91/02807 (Amgen) (=U.S. Pat. No. 5,158,875) discloses a method forrefolding IGF-I in the presence of a fused short positively chargedleader sequence, in which amino acids, such as lysine, arginine andhistidine are fused at the N-terminus of IGF-I. Inclusion bodies areisolated and solubilized with urea. In WO 93/11240 (Genentech) a methodfor refolding of insoluble and improperly folded IGF-I is describedinvolving solubilisation of inclusion bodies and refolding in a singlebuffer system.

U.S. Pat. No. 5,151,501 (American Cyanamid) discloses a process forsolubilization and naturation of somatropins (Growth hormones) bydispersing somatropin refracfile bodies in a solution containingsulfolane and thereafter dilution.

WO 9319084 ( Synergen) discloses a method for producing active IGF-I isclaimed, comprising the steps of expressing in prokaryotic cell, addinga first reducing agent, adding denaturing agent (e.g. urea), addingoxidizing agent (e.g. oxidized gluthatione or cystein) and adding asecond reducing agent (e.g. DTT, cystein etc.). Met-IGF-I is expressed.

WO 9506064 discloses a process for increasing the yield of correctrefolding of a polypeptide and in which a copper or manganese salt arepresent during the refolding step and WO 9506059 (Genentech) discloses amethod for isolation of cells by adding a phase-forming species to formmutilple aqueous phase.

SUMMARY OF THE INVENTION

We have now invented a novel, simplified method for the production ofcorrectly folded biological active recombinant protein or polypeptide.

Especially we refer to recombinant IGF-I after expression of Z- IGF-I.Reference is here given to EP 230 869, especially the examples.

With Escherichia coli expressing the hybrid protein Z-IGF-I, we haveachieved very high expression levels (up to 15 g/l fermentation).

Although the method here is described with IGF-I as the preferredpolypeptide, it can also be used for recombinant preparation of otherpolypeptides, when misfolded species and the overall yield of correctlyfolded polypeptide is rather low.

With our claimed method we can avoid the steps of mechanical disruptionof the cells and the isolation and washing of refractile bodies.

Our process is easier than the earlier described and gives a good yield.

The invention relates to a method for producing a correctly folded,biological active recombinant protein or polypeptide, comprising thesteps of

a) expression of the protein in prokaryotic cells,

b) harvest of the cells),

c) direct solubilization of the cells in a buffer at pH about 8 to 11,preferably about 8, with a chaotropic agent and a reducing agent, and

d) dilution with water and a diluent.

The protein could e.g. be IGF-I, IGF-II or GH.

When the protein is IGF-I the method preferably comprises the steps of

a) expression of an IGF-I-fusion protein in prokaryotic cell system,preferably E Coli,

b) harvest of the cells),

c) direct solubilization of the cells in a buffer at pH 8 to 11,preferably about 8, with a chaotropic agent and a reducing agents,

d) dilution with water and a diluent,

e) addition of a cleaving agent, and

f) purification to produce the biological active IGF-I.

The IGF-I-fusion protein is preferably a hybrid Z-IGF-I. The buffer instep c) could be e.g. Tris (Tris [hydroxymethyl]aminomethanehydrochloride) or glycine.

The chaotropic agent in step c) is preferably guanidine or urea andguanidine could be used in a concentration of 3-7M, preferably 5M.

The reducing agent in step c) could be e.g. DTT (DL-Dithiothreito) orcysteine and the diluent in step d) could be ethanol.

After step d), pH is preferably reduced below pH 6 and more preferablyto pH 3 or below.

For the preparation of IGF-I pH is preferably reduced to pH 3 or belowbetween steps d) and e).

A concentration step and a buffer exchange between steps d) and e), inwhich chromatography and/or ion-exchange preferably is used is alsoclaimed.

The cleaving agent in step e) in the preparation of IGF-I could behydroxylamine, an enzyme or any other cleaving agent.

The purification steps for the preparation of the pure protein orpolypeptide include e.g. cation exchange, RP-HPLC and/or hydrophobicinteraction Chromatography (HIC).

When IGF-I is produced as Z-IGF-I, IGF-I has a better solubility, whichmeans that we can have a higher concentration of reduced IGF-I insolution, giving a high amount of right folded IGF-I which is of mostimportance for an industrial method for the production of IGF-I.

EXAMPLES

The recombinant human IGF-I (rhIGF-I) used in the experiments wasproduced in E Coli according to the method described in EP 230 869,example VIII (but in a fermentor) including growing at 37° C. for 20hours.

Example 1 Solubilization

The cells were harvested after fermentation by centrifugation or crossflow filtration.

Thereafter the cells were dissolved in:

13 L cell solution, wet weight 498 g/L

5 mol/L guanidine-hydrochloride

97 mmol/L Tris-base

159 mmol/L Tris-HC1

2 mmol/L ethylene-dinitro-tetraacetic acid-disodiumsalt-dihydrate (EDTA)

4 mmol/L dithiothreitol (DDT)

Total volume: 16.9 L

The pH was kept at 8.1, the solubilization was run for 3 hours understirring at 150° C.

Refolding

The solubilization solution was diluted with 33.8 L 22.5% ethanolsolution, dilution factor 3.

The pH was kept at 8.1, under stirring at 15° C.

The refolding was stopped after 20 hours by addition of concentratedhydrochloric acid until the pH of the solution was <3.1.

RP-HPLC analysis of the concentration of Z-IGF-I in the refoldingsolution gave correctly folded 3.249 g ZIGF-I/L.

Example 2 Solubilization

The cells were harvested after fermentation by centrifugation or crossflow filtration.

Thereafter the cells were dissolved in:

154 mL cell solution, wet weight 450 g/L

4.5 mol/L guanidine-hydrochloride

400 mmol/L glycine

0.2% Tween 20

0.2 mmol/L ethylene-dinitro-tetraacetic acid-disodiumsalt-dihydrate(EDTA)

3 mmol/L dithiothreitol (DDT)

Total volume: 200 mL

The pH was kept at 10.0, the solubilization was run for 3 hours understirring at room temperature.

Refolding

The solubilization solution was diluted with 125 mL ethanol, 400 mLwater, 66.8 g guanidine-hydrochloride, dilution factor 4.

The pH was kept at 10.0, under stirring at room temperature.

The refolding was stopped after 20 hours by addition of concentratedhydrochloric acid until the pH of the solution was <3.1.

RP-HPLC analysis of the concentration of Z-IGF-I in the refoldingsolution gave correctly folded 1.38 g ZIGF-I/L.

Example 3 Solubilization

The cells were harvested after fermentation by centrifugation or crossflow filtration.

Thereafter the cells were dissolved in:

900 mL cell solution, wet weight 720 g/L

6 mol/L guanidine-hydrochloride

97 mmol/L Tris-base

159 mmol/L Tris-HC1

2 mmol/L ethylene-dinitro-tetraacetic acid-disodiumsalt-dihydrate (EDTA)

3 mmol/L dithiothreitol (DDT)

Total volume: 1.17 L

The pH was kept at 8.0, the solubilization was run for 3 hours understirring at 15° C.

Refolding

The solubilization solution was diluted with 547 mL ethanol 1786 mLwater, dilution factor 3.

Total volume: 3.6 L

The pH was kept at 8.1, under stirring at 15° C.

The refolding was stopped after 21 hours by addition of concentratedhydrochloric acid until the pH of the solution was <3.1.

RP-HPLC analysis of the concentration gave correctly folded 1.32 gZ-IGF-I/L.

Yield

Solubilization: 74% (of ZIGF-I from the fermentor)

Total, solubilization and refolding: 41%

Finally purified IGF-I was shown to have biological activity in e.g.chick embryo femore and RRA (Radio Receptor Assay).

Example 4 First Purification Step

A solution from the refolding step was diluted with WFI (Water forInjection), dilution factor 3 and clarified on a cross flow membranebefore applied on a cation exchanger (Pharmacia Biotech BPG 200/500,SP-Sepharose FF, 9.5 Liters, 0.33 m bed high).

The following buffers were used:

Step Buffer pH Equilibration 100 mM Citric acid/di-Na hydrogen phosphate3.0 Wash 1 50 mM Citric acid/di-Na hydrogen phosphate 2.9 Wash 2 100 mMdi-Na hydrogen- and Na-dihydrogen 5.9 phosphate Elution 100 mM Nahydrogen phosphate 7.5 Regeneration 500 mM NaOH Sanitation 100 mM NaOH

The column was washed with 1 Column volume (CV) WFI, the column wastreated with 3 CV of Equilibration buffer and the sample was hereafterapplied on the column.

The column was first washed with 3 CV of Wash 1 buffer and thereafterwith 4 CV Wash 2 buffer.

The product was eluted with 5 CV of elution buffer and the columntreated with 2 CV of Regeneration buffer and 2 CV of Sanitation buffer.

According to RP-HPLC the yield over the purification step was 98%correctly folded IGF-I.

Example 5 Cleavage Step

After the first purification step on a cation ion exchange column, thecleavage of Z from IGF-I was performed.

To a vessel was added: 40.0 Liters from the cation ion exchanger pool,1428 grams Sodium diphosphate and 4.0 Liters Hydrozylamine, 50% sol.

The pH was adjusted to 9.55 (with NaOH) and the temperature was kept at40° C.

After 3 hours the reaction was stopped by lowering the temperature to25° C. and adding 40.0 Liters concentrated Acetic acid (HAc) and 160Liters WFI.

The pH was adjusted to 3.30.

According to RP-HPLC the yield over this step was 84% of correctlyfolded, non oxidized IGF-I.

Example 6 Cleavage Step

After the first purification step on a cation ion exchange column, thecleavage of Z from IGF-I was performed.

To a vessel was added: 18.6 Liters from the cation ion exchange pool,664 grams Sodium diphosphate and 2.17 Liters Hydrozylamine, 50% sol.

The pH was adjusted to 9.5 (with HAc) and the temperature was kept at40° C.

After 3 hours the reaction was stopped by lowering the temperature to25° C. and adding 22.3 Liters concentrated HAc and 93 Liters WFI.

The pH was adjusted to 3.35.

According to RP-HPLC the yield over this step was 61% of correctlyfolded, non oxidized IGF-I.

What is claimed is:
 1. A method for producing a correctly folded,biologically active recombinant protein or polypeptide, comprising thesteps of a) expressing the protein or polypeptide in prokaryotic cells,b) after step a), harvesting the cells by a process comprisingcentrifugation or cross flow filtration, c) after step b), directlysolubilizing the cells in a buffer at pH of about 8 to 11 with achaotropic agent and a reducing agent to form a solution, and d) afterstep c), diluting the solution with water and a diluent, therebyobtaining the correctly folded, biologically active recombinant proteinor polypeptide, the method being conducted in the absence of mechanicaldisruption of the cells and in the absence of isolation and washing ofrefractile bodies from within the cells.
 2. A method according toclaim
 1. wherein the protein is IGF-I, IGF-II or GH.
 3. A methodaccording to claim 1, wherein the protein is IGF-I and comprising thesteps of a) expressing an IGF-I fusion protein in a prokaryotic cellsystem, b) after step a), harvesting the cells by a process comprisingcentrifugation or cross flow filtration, c) after step b), directlysolubilizing the cells in a buffer at pH of 8 to 11 with a chaotropicagent and a reducing agent to form a solution, d) after step c),diluting the solution with water and a diluent, thereby obtaining thecorrectly folded, biologically active recombinant protein orpolypeptide, e) adding an agent for cleaving IgG binding domain fromIGF-I, and f) purifying the IGF-I product of step e) to produce thebiologically active IGF-I.
 4. Method according to claim 1, wherein theprotein is hybrid Z-IGF-I.
 5. Method according to claim 1, wherein thebuffer in step c) comprises Tris or glycine.
 6. Method according toclaim 1, wherein the pH in step c) is about
 8. 7. Method according toclaim 1, wherein the chaotropic agent in step c) comprises guanidine orurea.
 8. Method according to claim 1, wherein the reducing agent in stepc) comprises DTT or cysteine.
 9. Method according to claim 1, whereinthe diluent in step d) comprises ethanol.
 10. Method according to claim1, wherein the pH is reduced after step d).
 11. Method according toclaim 3, wherein the pH is reduced to a pH of not greater than 3 betweensteps d) and e).
 12. Method according to claim 3, further comprising aconcentration step and a buffer exchange between steps d) and e). 13.Method according to claim 3, wherein the cleaving agent in step e)comprises hydroxylamine.
 14. A method according to claim 3, wherein thecell system comprises E. Coli.
 15. Method according to claim 7, whereinthe chaotropic agent comprises guanidine in a concentration of 3-7M. 16.A method according to claim 10, wherein the pH is reduced after step d)to a pH below
 6. 17. A method according to claim 10, wherein the pH isreduced after step d) to a pH not greater than
 3. 18. Method accordingto claim 12, wherein the concentration step employs at least one ofchromatography and ion-exchange.
 19. Method according to claim 18,wherein the purification step f) includes at least one of cationexchange, RP-HPLC and hydrophobic interaction Chromatography (HIC).